MembraneQuant Module 1.12 User's Guide 19 June 2009
Contents
1. Nucleus Settings Parameter settings of the nucleus You can adjust the threshold the smoothness and the erosion values You can also filter objects by size and shape Median filter Simple but fast filtering method it uses the median of adjacent pixel values A 3X3 blurring is recommended on an ordinary histo logy sample and when working on low quality samples too much noise on the image it is recommended to raise up the values Local Intensity Amplifier Amplificates the local maximums the area of which complies with the R radius filter Sets the intensity to the average cell size The value is 20 25 pixels on a sample taken with 20x magnification level zoom Threshold Sets the pixel intensity threshold values under the threshold will be defined as background The optimal value is between 20 and 30 on an average intensity slide Separate The pixel distance between two seeds which separates the spots if the given value is smaller than half of the seed distance The less the value the more it reduces the distance Filter Objects By Size Sets the size of the objects in um that MembraneQuant should detect as objects Excludes pixel groups that are too small or too large It enables you to filter the smaller nuclei of the inflamed part of the tissue Filter Objects By Shape Sets the shape of the objects that MembraneQuant should de tect as objects This filtering method marks the roundish2. 11 Launch the MembraneQuant algorithm by clicking Start in the Membrane win dow A Warning By using the same annotation and MISP file the program overwrites the previously saved CSV file If you want to create a different file locate an June 19 2009 3DHISTECH Ltd 29 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant other folder to save measurement data The process is fully automated you only need to click Close when Mem braneQuant has finished The application leaves the slide open If more than one slide is open when you launch the Wizard MembraneQuant always uses the active one Tip To view the existing annotations and measurements on the slide click INFO in the slide toolbar and then select Show Annotations or Show HQ Measurements from the shortcut menu 3 4 Running Batch Process in MembraneQuant In Image Analysis Modules menu you have the option to work in batch mode Select the slides you want MembraneQuant to work with and specify the parameters accord ing to which they should be analyzed 3 5 Saving MembraneQuant Results to the Slide MembraneQuant automatically saves the following data results to the slide e outline of the detected objects measured data morphometrical or colorimetrical data on the detected objects and area specific parameters 3 6 Exporting MembraneQuant Results in CSV Format Measurements from One Slide When you create the MIS
3. 6 Set the parameters If you enter new values into the text fields press Enter to refresh the diagram 35 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization 8 To create a snapshot of the histogram click Save 9 To exit Measurement Visualization click Close 4 3 Creating a Gallery 1 Click the E icon in the slide toolbar in MIRAX Viewer only in case no other data visualization window is open L Data Visualisation Data Selection Misp names Les MD_Modified 151328_18062009 E Annotations O Caption Freehand Annotation_Area LT Caption Rectangle Annotation_Area Layers Membranes v Mode Histogram Show O Scatter Plot Gallery Classification Galler 2 Select the name of measurement from the MISP names drop down menu in the Data Selection panel 3 Select the annotations that you want to display 4 Select the object layer that you want to display 5 Select Gallery and the press the Show button If you select Gallery button in the Mode function panel of the previously opened Data Visualization window then press Go The process steps can be seen in the title bar of the new window for example H 1 gt S 1 gt G 1 June 19 2009 3DHISTECH Ltd 36 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization 6jAvgCI 24 5507 ContourlOD 375 0255 Score 2 SF 0 6368 Copy T
4. fz ES 3DHISTECH 19 June 2009 MembraneQuant for MIRAX Viewer 1 12 User s Guide Contents Disclaimer ssh ne RS esis a sense me seen 4 Font types and E nl siel scence eee eee eee inai EEE EEE REEDS EEE E EEE E SEES SESE EH rr SEEDS EERE SEES E EEE 5 Terms and Tsis Te gis iii ia A ERA avast dvi ases amant eee sienne goss au ee 6 L About Membrane Quan tiamina das 7 1 1 MembraneQuant in the MIRAX product family e e ee e e e eee 7 1 2 Overview of Membrane ANalYSiS 0ooccccccconconnonnnnnncncnncnnconconcon corran nr narrar narrar nar EEE EEE 8 1 2 1 MembraneQuant algorithms sisi 8 1 2 2 Annotations Fields and Objects in MembraneQuant sese e e e ee e ee eee 8 W273 CCE yl 10 1 254 MISP RES a Rage ann MANU A soda dar tiens rem diner enw 10 1 2 5 Using a MISP file on other slides 10 1 2 6 MembraneQuant Measurements eee ee enn EEE ERAS nnn EES 10 1 2 7 Measurement Export in CSV Format sise 11 1 3 Overview of Measurement Visualization sisi 11 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 nr een been ve RH aR de dns ne Nes iii 13 2 1 Prerequisites for the MIRAX Viewer Installation 13 2 I B LICENSING coria decd mean eee ne eme A 13 2 2 Installing MIRAX Viewer and Its Modules 13 3 Working with MembraneQuaNt incio miii A ease KaR 9 ea baad NNO eerie 21 3 1 Before YOU Start ce E TART RENS b
5. 1 CNO 1 NO 100 2 NO 2 NO 100 3 NO 3 NO 100 MA Overall mask area in mm Total area of all detected objects in each layer MAO Mask area of negative Membrane MA 1 Mask area of 1 Membrane MA 2 Mask area of 2 Membrane MA 3 Mask area of 3 Membrane NO Number of detected objects Total number of each detected object in each layer NOO Number of negative Membrane NO 1 Number of 1 Membrane NO 2 Number of 2 Membrane NO 3 Number of 3 Membrane rMA MA FA 100 relative mask object area in rMAPos CMAO 1 MAO 2 MA 3 MA 100 rNOPos CNO 1 NO 2 NO 3 NO 100 June 19 2009 3DHISTECH Ltd 43 43
6. 12 User s Guide U H 1 gt 8 1 Joy SF 650 690 6 0 680 6t 0 Statistics Overal 52 X Axis Min Value Max Value Linear Mode O Gallery 220 225 230 235 240 245 250 255 260 265 270 275 280 285 Area um 2 Copy To Clipboard Parameter Area Histogram O Scatter Plot O CSV file export B ral e Qs Selection count 10 Percent 192 v Auto Refresh Y Axis iv Parameter SF S Min Value M AutoScale Y AutoScale Max Value Logarithmic Linear Logarithmic Gate Color CJ un Ce Auto Refresh Automatically updates the histogram when you change the settings for the parent form Statistics Overall it shows the quantitative data of the objects X Axis Offers various parameters for the X axis Y Axis Offers various parameters for the Y axis Gate Chooses an interval within the examined range on X axis You can position the scatter plot gates by clicking on the arrow Mode By using Gate function the program either creates new histogram new scatter plot shows gallery or opens a new form where the user can save CSV files but only in the selec ted interval By not using Gate function the program uses all the above mentioned features but for the whole data range June 19 2009 7 Press Back to get to the parent window 3DHISTECH Ltd 4 Working with Measurement Visualization
7. Applications By downloading or installing the software 3DHISTECH s Applications the Software the Licensee accepts you accept the following terms and conditions Van amvaa tn he hond hrr the tomo and annditinns af thie End Tear Tirence ae If you accept the terms of the agreement select the first option below You must accept the agreement to install 3DHISTECH s Applications Click Next to continue 3DHISTECH Ltd 6 Select the components you want to install 2 3DHISTECH s Applications Setup le S gt Choose Components Choose which features of 3DHISTECH s Applications you want to SDHISTECH install Check the components you want to install and uncheck the components you don t want to install Click Next to continue Description Select components to install Space required 45 8MB arran HistoQuant NuclearQuant MembraneQuant FISHQuant ImmunoScreener TMA La Position your mouse a component to see its description SDHISTECH Ltd June 19 2009 3DHISTECH Ltd 15 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 7 If not previously done you have to install the prerequisites of the installation 1 3DHISTECH s Applications Setup La m X Prerequisites Review the prerequisites of the installation 3DHISTECH Status NET Framework 2 0 Waiting For i
8. User s Guide Disclaimer Copyright 2001 2009 3DHISTECH Ltd All rights reserved The following patents are pending for 3DHISTECH products EP07733864 8 US 12 301 781 EP07713006 0 US 12 281 566 PI0708550 8 20070007642 2 US 2008 0306964 A1 HKO7113109 4 EPO5702806 0 3DHISTECH Ltd is not liable for damage of whatever nature including but not limited to general or specific damage indirect damage consequential damage or incidental damage including the results of the analysis of the digitized slides for example change of health status related to erroneous diagnosis from the digitalized slide s that stems from or is associated with use of Product digitalized slides quality of staining quality of stained slides quality of used method of staining Certain legal systems do not allow the limitation or exclusion of accidental damage This restriction therefore may not apply in your case 3DHISTECH Ltd assumes no responsibility for the functionality and fault free condition of your application programs Workflows VBA macros Commander scripts CAUTION For research and education uses only not for use in diagnostic procedures This product has not been approved or cleared as a medical device by the U S Food and Drug Administration nor any other regulatory authority The data and images obtained or viewed using this product are not intended for clinical or diagnostic use Third party disclaimer LibTIFF TIFF Library and Ut
9. free hand and cover the area with the annotation For information on navigating and zooming on the slide see section Viewing slides in the MIRAX Viewer 1 12 User s Manual Inthe Create or Edit Annotation window add a title to the annotation enter a comment and determine the background color of the title and the outline color of the annotation You can either select from predetermined colors or you can define custom colors Only captions are exported to the CSV output file Annotation descriptions are vis ible only in MIRAX Viewer SS BIDAR la Uno JA EA EG 73 107 EE Magniication 16 65 Progress WIN 3 Repeat step 2 for each area on which you want to run the algorithm 3 3 Running the Algorithm on a Single Slide Open the slide in MIRAX Viewer 1 In the main toolbar click the 4 icon and select MembraneQuant from the Image Analysis Modules June 19 2009 3DHISTECH Ltd 22 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant EX Image analysis modules Wok Slide G slides Membran10301 07_HER2 General applications HistoQuant is an image segmentation solution for general purposes based on color brightfield or wavelength fluorescent information IHC applications NuclearQuant detects and separates nuclei on immunolabeled slides Le ER PR Ki67 MembraneQuant analyses cellmembrane positivity on immunolabeled slides Le HER 2 EGFR ep Fluorescen
10. other algorithms on sev eral annotations by selecting all the annotations that you need when you start the al gorithm Important MembraneQuant algorithms utilize the computer s hardware mainly the CPU and the memory The speed of the MembraneQuant evaluation de pends on the hardware configuration and performance the size and the number of the measured annotations and the quantity of detected ob jects MembraneQuant algorithm runs only on Brightfield slides To ensure accurate results and to reduce computation time e Make the training annotation as small as possible The annotated areas should not be larger than 300x300 um 1 1 magnification When you are creating a free hand annotation follow the outline of the area as closely as possible e Select the annotation area appropriately for example select an area that is rich in tumor cells instead of connective tissue y If you need to make measurements on the entire specimen simply select the whole slide 3 2 Creating Annotation for MembraneQuant 1 Open the slide in MIRAX Viewer For more information on how to open slides in MIRAX Viewer see section Working with slides in MIRAX Viewer in the MIRAX Viewer 1 12 User s Guide June 19 2009 3DHISTECH Ltd 21 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant 2 Create the annotation on the area of your interest Select the annotation type rectangle or
11. parameters of the detection in the Membrane Analysis Setup window create a Color Deconvolution Profile 3 3 1 Setting up a Color Deconvolution Profile 5 Click Setup to specify Color Deconvolution parameters O Important There are four preset CCC files according to different camera sensitivities You can set the software to your automated slide stainer or labor protocol with this ap plication When assigning manual settings it is recommended to take random intensity samples from the preview screen of the most dense areas of the chromogen If you get the desired result in the chromogen preview and the parameters are set cor rectly modify the color settings of counterstain Marking with chromogen can result in a strong background color please take this into account when sampling counterstain It is recommended to take samples from the background area of the image Note The software automatically refreshes the displayed chromogen and counterstain preview images according to your settings In the Color Deconvolution Setup window you see a magnified image of the slide where a preview of the original can be switched on and off by pressing Show Preview and the RGB color spectrum of the marker colors can be adjusted O Counterstain Hematoxilin X a C d e TELE 4 PL D E k L d LA x k June 19 2009 3DHISTECH Ltd 25 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with Mem
12. you enter new values into the text fields press Enter to refresh the diagram June 19 2009 3DHISTECH Ltd 32 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Lui Joy Piece Gate 50 100 Copy To Clipboard Statistics Overall 292 X Axis Min Value Max Value Mode Histogram O Scatter Plot O Gallery Gated 52 Ratio 17 8 AVG Gated 250 STD Gated 20 16 CV Gated 8 06 Parameter eas T T T T T T T T gt 150 200 250 300 350 400 450 500 550 Area um 2 M Auto Refresh AVG 264 6 STD 141 93 CV 53 64 Number Of Classes 25 AutoScale Linear Logarithmic Gate O CSV file export Lower Value 218 7071 Use Gate Upper Value 288 272 Auto Refresh Automatically updates the histogram when you change the settings for the parent form Statistics Overall shows the quantitative data of the ob jects X Axis Offers various parameters for the X axis When you export the results into a CSV file there is an explanation for the abbreviations of the parameter names Gate Chooses an interval within the examined range on X axis Mode By using Gate function the program either creates new histogram new scatter plot shows gallery or opens a new form where the user can save CSV files but only for the se lected interval By not using Gate function the progra
13. 1 12 User s Guide ImmunoScreener Module for MIRAX Viewer 1 12 User s Guide MIRAX SCAN User s Guide Image processing and analysis l http books google com books id kvWkKgG6iPXMC amp dqO June 19 2009 3DHISTECH Ltd 41 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization Index A Anotaciones 2 6 8 12 21 24 27 29 32 34 36 38 40 44 B Bate PROCESS ta 2 30 C CCC Re ne dt radin da OE ee a ae Co er done Mons 2 6 10 25 26 CNEOMOQEN RAS asie A A sein inerte A AA T 25 Classification Gallery lt sss sad an 9 S ST ses ives is dis A Re 2 38 Color DeconVOlUtiON TTT 2 6 8 10 25 28 Colorimetrical data uses A Re sn an wah may ca bel es oies nee 30 Computational i ee dt 21 COUNTEFSTAIME 55 semis ea ER EERE daw tive aden sien deen meus AA DA AA 25 CSV ores A E Sa isis wen ee vee ene eee eee manne ne cea 2 6 8 11 22 24 29 31 33 35 39 F ER 2 5 6 8 12 28 32 35 37 40 Sasha seen nr eme ear seras einem ste oes steaks nade ease 40 G ET sean ce esse sas tatin ne ee eds de ares 2 8 9 33 35 38 Cie a ee AT E rere reer crc rer eee reer ee er cre ee eer rere eer creer ee reer ee cre ee Tere 33 35 H ES LL 2 8 32 33 35 37 L ETS ii LS 2 6 9 11 24 29 32 34 36 38 44 Local Intensity Amplifier a E EERE EEE EEE E rr 27 Longitudinal StUGICS tissctrectesctvecttaennadd pe dador AI ida 11 M MeasuremMment s issssssssanessateseneenescermenseiman
14. MIRAX microscopes for example MIRAX SCAN For more details see MIRAX SCAN User s Guide A digitized slide consists of an MRXS file and a folder with the same name as the MRXS file June 19 2009 3DHISTECH Ltd 7 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 About MembraneQuant 2 Digitized slides are stored on a computer You can also use a dedicated server for example MIRAX Server for storing the MIRAX slides 3 Slides digitized with a MIRAX microscope can be viewed in MIRAX Viewer If the slide is stored on a MIRAX Server it is opened through the Teleconsultation module Local slides are opened directly with MIRAX Viewer 4 MembraneQuant allows you to perform measurements on digital slides and to export measurements in CSV format 5 Measurement Visualization displays the MembraneQuant data as a scatter plot a histogram an image gallery or a classification gallery 6 Digital slides including slides that store MembraneQuant measurements can be shared via PathoNet www pathonet com They can also be used in E School as training and or exam materials 1 2 Overview of Membrane Analysis You can perform two types of tasks in MembraneQuant set algorithms then run them on a slide or a set of slides view the measurement results with the Measurement Visualization module save the measurement annotations with the slide or export the measurement res ults to CSV file s create a report based on your saved m
15. P file MembraneQuant exports the results to a CSV file If you need measurements from this slide provide a new file name If you create several ayers in the annotation with the MembraneQuant algorithm the data for each layer can be exported into a separate CSV file Furthermore a cumulative CSV file is also exported with the ayer data Note CSV file names are generated automatically from the combination of names of MISP slide and annotation June 19 2009 3DHISTECH Ltd 30 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant 1 Slide information 2 Name of the annotation s that you selected for the CSV export If you selected several annotations on the same slide for the CSV export the CSV file lists them at the top of the table the object IDs of the annotations are listed one after the other at the bottom section of the table 3 Objects Each object that the MembraneQuant algorithm found in the annotation is shown in a sep arate row 4 Parameters Each parameter is shown in a separate column 5 Measurement Each measurement the value of a parameter in an object is shown in a separate cell 3 7 Exporting MembraneQuant Results to a Cumulative CSV File When you create the MISP file MembraneQuant exports the results to a CSV file If you require measurements from several slides to create a cumulative database select the CSV file to which MembraneQuant appends tw
16. ave Automatically saves all changes Note that it decelerates the program Border size slider Increases decreases the size of object bor ders in the right panel Zoom factor slider Zooms in out on the objects in the right panel Toggle mode You can select objects with the help of your mouse in the right panel You can rescore ob jects using right click Tooltip Moving cursor over an object shows the basic information of the given object View You can select among normal horizontal or vertical view of the objects in the right panel June 19 2009 3DHISTECH Ltd 28 29 31 El E 41 42 43 39 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization By right clicking on a selected object or selected objects you can rescore or delete them select all or none and or show their annotations ReScore Delete Show annotation Select all Select none The Field Parameters window lists parameter names and values of that specific annota tion Scroll down or change window size to see the whole list Field Parameters ES Caption Freehand Annotation_ Area Value 5 093E 2 3 0343E 2 1341 M 59577641 NO E 6954E 3 Hscore 203 812316715543 June 19 2009 3DHISTECH Ltd 40 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization Related documents MIRAX Viewer
17. braneQuant Load You can load predefined Color Deconvolution Profiles CCC files Save You can save your settings to Color Deconvolution Profiles Show Preview A preview window opens where you can select the area of your interest You can position the image in the left side window by dragging You can get color samples from the image on the left Color boxes Left click on the boxes to adjust the RGB color spectrum if neces sary with the help of the slides or type in the required values press Enter to refresh Right click on the boxes to reset the sampled color to black Note The software continuously refreshes the two preview images on the right side of the window during sampling and also note that sampling has effect on both preview im ages 3 3 2 Setting Detection Parameters 6 After saving a Color Deconvolution Profile click Close and define the paramet ers of the algorithm in the Membrane Detection tab on the left side of the Settings Preview window Membrane Detection Scoring Show results 14 Image Original v Cell spots None v CL Membrane U Nucleus il Membrane Smooth 1 No Nucleus Crossing Merge membrane spots When border intensity gt 200 Z Weakness Length J y a Area pr lt v 100 00 Filter lt Average Intensity lt 20 Y J 255 2 lt Area pe lt 30 00 g J 800 00 i Nucleu
18. e Cumulative CSV One cumulative file will be N One cumulative file will be created created for each ayer of objects detected e Ifyou enter a new file name the CSV export will contain all data in the project If you select an existing CSV file only the summary data will be added to the existing file Use this option to create a cumulative file for example for longitudinal studies or to follow up a pa tient s history Y Tips y Check the documentation of your statistical or spreadsheet application to ensure that the CSV file exported from MembraneQuant is compatible with that application Also make sure that the regional settings of the operating system MIRAX Viewer and the post processing application are the same Otherwise sep arators comma semicolon etc used during the CSV export will not be recognized in the statistical or spreadsheet application 1 3 Overview of Measurement Visualization Measurement Visualization is an extension to MembraneQuant which allows you to create a graphical representation of measurement results obtained from the Mem braneQuant algorithm run on a slide or set of slides grouped into an ImmunoScreener project For more information on ImmunoScreener see ImmunoScreener Module for MIRAX Viewer 1 12 User s Guide June 19 2009 3DHISTECH Ltd 11 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 About MembraneQuant Measurement Visualization works only with
19. e MISP on other slides from the same batch for example on slides that are from the same specimen without modifications You can also use the same algorithm on slides from other specimens with the same staining You might need to adjust the profile to ensure that differences in the specimen or stain quality do not affect the result 1 2 6 MembraneQuant Measurements MembraneQuant creates the following types of measurement e object specific measurement related to an object e is related to an object for example its area For more information see Ap pendix A1 field specific measurement related to an annotation sums up object data for example the total area of objects of the same layer in an annotation For more information see Appendix A2 conveys information about the annotation for example its total area For more information see Appendix A2 June 19 2009 3DHISTECH Ltd 10 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 2 7 Measurement Export in CSV Format 1 About MembraneQuant You can export MembraneQuant measurements as CSV files Both field level and ob ject level measurements are exported You can choose between the following options Object Specific CSV Saves the parameter values for each object in the annotations that you select for the CSV export If you use an existing file name the old file will be overwritten Cumulative CSV Single File Cumulative CSV Multi Fil
20. e a TR da ve Naa REE RENK RSS EaR eves deere 21 3 2 Creating Annotation for Membrane Analyzer ssssss 21 3 3 Running the Algorithm on a Single Slide 22 3 3 1 Setting up a Color Deconvolution Profile ss 25 3 3 2 Setting Detection Parameters sssssssssisssssessesceseseeneeseesesenss 26 3 329 HOW to Set the SCOPING seve sess dices sone seasons Ai 28 3 4 Running Batch Process in MembraneQuant sssssss 30 3 5 Saving MembraneQuant Results to the Slide ss 30 3 6 Exporting MembraneQuant Results in CSV Format Measurements from One Slide 30 3 7 Exporting MembraneQuant Results to a Cumulative CSV File 31 4 Working with Measurement Visualization rr enn nnn nents 32 4 1 Creating a iMiStO Grams vacsicsyesceusi a os 32 4 2 Creating a Scatter PlOtd crea aT EES A visserie einstein teen aia wE 34 4 3 Creating a Gallery ivan io AA da a A 36 4 4 Creating a Classification Gallery sise 38 Related docente eva 41 ING OX bese deasesieosesesdseiasesaavsavvedoteesevesesvehotaes vednesdsesieesasyiantias vs isdamsenboescrs E avt 42 June 19 2009 3DHISTECH Ltd 2 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Appendix A MembraneQuant c sciiiiecsde ia Sa 29522 a e lavada teas Ra asada adie cada 43 A 1 Object specific parameters siemens 43 A 2 Summing parameters for an area 43 June 19 2009 3DHISTECH Ltd 3 43 MembraneQuant for MIRAX Viewer 1 12
21. easurements 1 2 1 MembraneQuant algorithms MembraneQuant is a membrane analysis application with color deconvolution profile settings You can set the parameters of smoothness and merger you can also filter the areas by size The information necessary for the algorithm is not saved on the slide but in a MIRAX Image Segmentation Profile MISP file which can be used later to evaluate other annotations on the same slide or on other slides Note Since MembraneQuant enables you to make measurements on the slides and di gitalized image processing can cause loss of quality please always make sure that the measurements are acceptable to you Measurement values are optimized by default set parameters manually only if the result is not satisfactory to you 1 2 2 Annotations Fields and Objects in MembraneQuant MembraneQuant uses annotations If you want to perform measurements on the entire digitized area of the slide you need an annotation that covers the entire area You can use any of the annotations created in MIRAX Viewer including annotations containing MembraneQuant measurements June 19 2009 3DHISTECH Ltd 8 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 About MembraneQuant L Tips You can add a caption and a description to an annotation If you want to see a piece of information for example a short comment to be visible in the gallery that you can export from MembraneQuant add it to the cap tion Only the annotati
22. esentation exe SDHISTECH Ltd 9 After completing the installation of MIRAX Viewer and its modules click Finish to close the wizard June 19 2009 Completing the 3DHISTECH s Applications Setup Wizard 3DHISTECH s Applications have been installed on your computer Click Finish to close this wizard Cancel 3DHISTECH Ltd 17 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and 10 Measurement Visualization Bundled with MIRAX Viewer 1 12 If you have an older or newer version of the applications you have to uninstall them first Version check Check computer for installed versions 3DHISTECH June 19 2009 Preuninstall Installed 3DHISTECH s Applications version 1 12 37 0 You must remove this apllication first To do that press Uninstall button 3DHISTECH Ltd Welcome to the 3DHISTECH s Applications Uninstall Wizard This wizard will quide you through the uninstallation of 3DHISTECH s Applications Before starting the uninstallation make sure 3DHISTECH s Applications are not running Click Next to continue 3DHISTECH Ltd 18 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 12 The wizard automatically detects the folder of the previously installed applica tions Click Uninstall to proceed 5 3DHISTECH s Applications Uninsta
23. ilities 3DHISTECH Ltd is not liable for damage of whatever nature including but not limited to general or specific damage indirect damage consequential damage or incidental damage including damage resulting from lost profits interruption of business loss of business information pecuniary loss or similar damage that stems from or is associated with the incorporated part of this Product that is made by Silicon Graphics Inc THE SOFTWARE IS PROVIDED AS IS AND WITHOUT WARRANTY OF ANY KIND EXPRESSED IMPLIED OR OTHERWISE INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FOR ANY SPECIAL INCIDENTAL INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE DATA OR PROFITS WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE AND ON ANY THEORY OF LIABILITY ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE Further information For the latest information on 3DHISTECH products and services visit http www 3dhistech com June 19 2009 3DHISTECH Ltd 4 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Font types and symbols Example Abbreviation or term that is explained in the section Terms and Abbrevi ations Example Words or characters that appear on the screen These include field names screen titles pushbuttons and menu
24. in Indicates the allowed deviation in intensity tensity June 19 2009 3DHISTECH Ltd 28 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant Up down left right Moves the annotation up down left and right 9 Save your settings and close the Membrane Scoring Setup form by clicking Ok 10 optional You can export Membrane Analysis measurements as CSV files by specifying the Export Settings Specify where the CSV file s should be saved a gt Q Membrane G slides Membran 10301 Modify Membrane Image Segmentation Profile Definition Profile Setup Annotation Selection Process Annotation Area Process whole Slide Available Selected Caption Rectangle Annotation Caption Freehand Annotation Export Settings Save Region measurements Save Cumulative measurements Save to single CSV Membrane G slides Membran 10301 07_HER2_I Save Region measurements MS Excel OpenOffice The measurements will be exported to a CSV file that you can open and further edit in any spreadsheet software for example ments Save Cumulative measure Cumulative measurements will be exported to CSV files A cumu lative file will be created for each detected ayer Save to single CSV The measurements will be exported to a single CSV file This file will contain all the parameters of the ayers
25. ll Wa Uninstall 3DHISTECH s Applications Remove 3DHISTECH s Applications from your computer 3DHISTECH 3DHISTECH s Applications will be uninstalled from the Following Folder Click Uninstall to start the uninstallation Uninstalling From C Program Files 3DHISTECHIMIRAX Viewer 3DHISTECH Ltd 13 When the uninstallation of MIRAX Viewer and its modules is complete click Fin ish to close the wizard 2 3DHISTECH s Applications Uninstall a o X Completing the 3DHISTECH s Applications Uninstall Wizard 3DHISTECH s Applications have been uninstalled from your computer Click Finish to close this wizard June 19 2009 3DHISTECH Ltd 19 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 14 After the previously installed applications are removed click Next to launch the installation wizard 3DHISTECH s Applications Setup Version check Check computer for installed versions 3DHISTECH June 19 2009 Preuninstall Uninstalled 3DHISTECH s Applications version 1 12 37 0 A Uninstall To continue the installation click the Next button 3DHISTECH Ltd 3DHISTECH Ltd 20 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant 3 Working with MembraneQuant 3 1 Before you start MembraneQuant works on selected annotations You can run
26. m uses all the above mentioned features but for the whole data range 7 Press Back to get to th e parent window 8 To create a snapshot of the histogram click Save June 19 2009 3DHISTECH Ltd 4 Working with Measurement Visualization 33 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization 9 To exit Measurement Visualization click Close 4 2 Creating a Scatter Plot 1 Click the ta icon in the slide toolbar in the MIRAX Viewer only if no other data 4 5 visualization window is open L Data Visualisation Data Selection _Misp names gt MD_Modified 151328_18062009 v Annotations O Caption Freehand Annotation_Area C Caption Rectangle Annotation_Area Layers Membranes v Mode Histogram Scatter Plot Gallery Classification Gallery Select the name of measurement previously saved to the slide from the MISP names drop down menu in the Data Selection panel Select the annotations that you want to display Select the object layer that you want to display Select Scatter Plot and the press the Show button If you select Scatter Plot button in the Mode panel of the previously opened Data Visualization window then press Go The process steps can be seen in the title bar of the new window for example H 1 gt S 1 June 19 2009 3DHISTECH Ltd 34 43 MembraneQuant for MIRAX Viewer 1
27. mesnantteeiese reset 2 6 13 21 24 29 32 34 36 39 M dian li ii A A A IA A AAA A da 27 MIS Puna adas 2 6 8 10 29 32 34 36 38 MRXS ir AD 6 7 R ROSCA A OT 38 40 e tant entente anse med Nb ete d ni s nat 25 26 S le ade e HHHH 2 8 32 35 37 SEMEN ON iran 6 8 10 Segmentation PrOfil s 2 45 o80ess8 donne nes ri ri io oi a 6 8 T TelecoNSUI AN iii A A A 8 21 V VittUal Slid escric acre aE E E E E kop EAN A tou hens 9 June 19 2009 3DHISTECH Ltd 42 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization Appendix A MembraneQuant A 1 Object specific parameters Parameter Description name Area Area of the detected object in m BInt B intensity of the detected object GInt G intensity of the detected object GrayInt Gray intensity of the detected object IF_C1 Integrated fluorescence in channel 1 IF_C2 Integrated fluorescence in channel 2 IF_C3 Integrated fluorescence in channel 3 LDia Longest diameter of the detected object in um Peri Perimeter of the detected object in um RInt R intensity of the detected object SDi Shortest diameter of the detected object in um SF Shape factor of the detected object A 2 Summing parameters for an area Parameter Description name FA Overall annotation area in mm fNO NO FA frequency of detected objects per um HSCORE
28. n panel Select the annotations that you want to display Select the object layer that you want to display Select Gallery and the press the Show button You can group your objects in accordance with their score values You can revalidate and rescore your object On the right side panel you can move cell nuclei from one group to the other On the left panel you can view the selected cell nuclei June 19 2009 3DHISTECH Ltd 38 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Y Slideview V Outline FM Boundary O Auto save 1 5003E 0 4 Working with Measurement Visualization Border size J D Toggle mode Tooltip Horizontalview a Zoom Factor __ Y Field parametres 8 5 8 aja ajaja ee 137 182 1 184 186 187 188 SISISIRIR SISIR r 83 238 344 393 396 397 412 419 439 444 451 452 E im asf sss 5155 455 466 467 501 509 516 527 528 531 532 535 AA ZEEL 545 546 562 569 600 603 629 635 636 588 592 2 essuie me 8 aj ajjaja aleje 50 S 18 1162 2 2089E 2 1 7102E 3 238 037865748709 21 22 K 24 25 26 27 El El El El El El El 33 34 35 36 37 38 39 Slideview Shows the selected slide Outline Shows the group specific outline Boundary Shows the number of the selected objects Export CSV Exports the measurements to a CSV file Save to slide Saves the changes to the slides Auto s
29. names paths or options Keys on the keyboard For example function keys such as F11 or the Ctri O key combination Example Cross references to other documents Warning Indicates that you need an additional module to use the feature or that there are prerequisites for the task or that special care should be taken when using certain feature of the device An important piece of information or a recommendation The application will work if you choose not to follow the recommendation but its perform ance might be less than optimal Tip A Y A suggestion about using the application in some other way or to some in teresting purpose June 19 2009 3DHISTECH Ltd 5 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Terms and abbreviations Annotation A particular detail of a digitized slide MembraneQuant can perform measure ments on annotations CCC The Color Deconvolution setting is stored in a ccc file CSV format Comma separated value Digital slide Slide digitized with a MIRAX scanner microscope for example MIRAX SCAN Consists of an MRXS file and a folder with the same name as the MRXS file Field In MembraneQuant an annotation In ImmunoScreener a grid cell Layer A group of cell compartments matching the same stain and size criteria Measurement MembraneQuant can perform two types of measurements e field specific related to the annotation s objec
30. ng MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 3DHISTECH s Applications Setup Welcome to the Setup Wizard of 3DHISTECH s Applications This wizard will guide you through the installation of 3DHISTECH s Applications It is recommended that you close all other applications before starting Setup This will make it possible to update relevant system Files without having to reboot your computer Click Next to continue Cancel 3 By default the installation folder is the following C Program files 3DHISTECH MIRAX Viewer 4 Set the license mode If necessary select install with HASP dongle otherwise select a license file 5 3DHISTECH s Applications Setup Joe License Settings Set license mode 3DHISTECH License Mode O Install with HASP dongle C Program Files 3DHISTECHIMIRAX Viewer MView lic SDHISTECH Ltd June 19 2009 3DHISTECH Ltd 14 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 5 Accept the End User License Agreement and click Next 1 3DHISTECH s Applications Setup L O Ey License Agreement Please review the license terms before installing 3DHISTECH s spiisTecn Applications Press Page Down to see the rest of the agreement General Terms and Conditions of End User License Agreement of the License La for 3DHISTECH s
31. nstall Microsoft Visual C Redistributable Waiting For install Intel Performance Primitives 5 2 Waiting For install Install the required components to continue SDHISTECH Ltd 8 Click Install to continue with the installation of the selected components S 3DHISTECH s Applications Setup Ce Oleg Prerequisites Review the prerequisites of the installation 3DHISTECH Status NET Framework 2 0 Installed Microsoft Visual C Redistributable Installed Intel Performance Primitives 5 2 Installed All of required components are installed To continue click Install button SDHISTEGH Ltd June 19 2009 3DHISTECH Ltd 16 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 A progress bar and an information box is displayed showing the status of the installation procedure Installing Please wait while 3DHISTECH s Applications have been installed 3DHISTECH Extract Extract Extract Extract Extract libquide40 dll 100 Extract Extract Extract Extract Extract Extract Data_presentation dll 100 Extract Extract Data_presentation exe cxcore097 dil 100 extsOD1 dll 100 highqui097 dll 100 trs dll 100 Mirax System dll 100 Mirax YiewerObject dil 100 Mirax StandardControls dll 100 AxMrxVisObLib dll 100 Mrx isObLib dll 100 Data_pr
32. nuclei of the connective tissue cells distinguished from the epidermal cells that have high shape factor Value ranges from 0 1 where 1 marks the round nuclei June 19 2009 3DHISTECH Ltd 3 Working with MembraneQuant 27 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant 3 3 3 How to set the Scoring 7 Select Scoring tab and set the membrane scoring setup 8 In the Scoring tab you can set Average Intensity values for the nuclei marked with 1 2 and 3 Automatic scoring can be set based on pixel intensity values When defining scoring values please take the previous settings and results of Color Deconvo lution into account Color Deconvolution Membrane Detection Scoring r Cell score e 0 all nuclei without membrane 1 all detected membranes if not 2 or 3 Where membrane intensity Le 2 average lt 180 standard deviation lt 30 S Where membrane intensity 3 average lt 150 Z standard deviation lt 20 Field score YM 0 None of below b 1 1 2 43 are gt ei 2 2 43 are gt sel 3 3 are gt Cell score Scores the cell membranes based on their intensity Field score Scores the whole field Average membrane intens Scores an area based on the average intensity ity Std Dev of membrane
33. o Clipboard Auto Refresh Border size L Zoom Factor J 0 94 Auto Refresh Automatically updates the histogram when you change the settings for the parent form Border size Sets the size of the area around the object Zoom factor Sets the zoom rate of the images You can determine the zoom value by entering the ex act rate in the text field If you move the cursor over an image an info box appears conveying all the ne cessary data regarding that image 6 Press Back to get to the parent window 7 To export the gallery as an image click Save 8 To exit Measurement Visualization click Close Tip If you select an image in the gallery it will be highlighted in the scatter plot as well June 19 2009 3DHISTECH Ltd 37 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization 4 4 Creating a Classification Gallery 1 Click the ka icon in the slide toolbar in MIRAX Viewer only in case no other 4 5 data visualization window is open 1 Data Visualisation Data Selection Misp names MD_Modified 151328 18062009 x Annotations O Caption Freehand Annotation_Area C Caption Rectangle Annotation_Area Layers Membranes v Mode Histogram Scatter Plot show Gallery Classification Gallery Close Select the name of measurement from the MISP names drop down menu in the Data Selectio
34. o rows 1 Slide information 2 The ayer information is visible in the parameter names in the column headers 3 Parameters that you selected for the MembraneQuant algorithm and for which measure ment data is available for CSV export June 19 2009 3DHISTECH Ltd 31 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 4 Working with Measurement Visualization 4 Working with Measurement Visualization The Measurement Visualization module for MIRAX Viewer creates a scatter plot or a histo gram from the MembraneQuant measurements 4 1 Creating a Histogram 1 Click the a icon in the slide toolbar in the MIRAX Viewer a ame L Data Visualisation Data Selection Misp names MD_Modified 151328_18062009 Annotations O Caption Freehand Annotation_ rea C Caption Rectangle Annotation_Area Layers Membranes we Mode Histogram Sho Scatter Plot ad Gallery Classification Galler S s 2 Select the name of measurement previously saved to the slide from the MISP names drop down menu in the Data Selection panel 3 Select the annotations you want to display By clicking the right side checkbox above the Annotations window you can select all the annotations To remove selections click the checkbox to the left 4 Select the object layer that you want to display from the Layer drop down menu 5 Select Histogram and the press Show button 6 Set the parameters If
35. object level measurements which means that if you select three annotations all object data are summed up but there is no in formation regarding how many data items derive and from which fields June 19 2009 3DHISTECH Ltd 12 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 2 Installing MembraneQuant and Measurement Visualization Bundled with MIRAX Viewer 1 12 MembraneQuant and Measurement Visualization are not stand alone applications you need MIRAX Viewer to run them Therefore you have to install MIRAX Viewer before installing the application 2 1 Prerequisites for the MIRAX Viewer Installation Prerequisites Description Hardware Minimum Pentium P4 3GHz 1GB RAM Recommended Pentium C2D DualCore 2 GHz 2GB RAM Operating system Microsoft Windows XP Disk space MIRAX Viewer 1 12 bundled with the MembraneQuant and Meas urement Visualization modules 1 12 Minimum 110 MB 2 1 1 Licensing You receive a product specific license when purchasing any of the 3DHISTECH applica tions 2 2 Installing MIRAX Viewer and Its Modules 1 Launch the 3DHISTECH_Apps exe file that you received on a 3DHISTECH in stallation CD or downloaded in a compressed file 2 Follow the instructions in the installation wizard June 19 2009 3DHISTECH Ltd 13 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 2 Installi
36. on caption is exported the description is not Create unique captions to make annotations distinguishable thus the measurements can be easily identifiable The following image shows a virtual slide treated with IHC stains There are two annota tions on the slide these are possible targets for MembraneQuant analyses fo rat DI So ara o La Cin CN EE ECS EE COX EU EC EE Maoriication 16 65 Progress EU At BER LA p pa AGE Z any Annotation types 1 field user defined annotation rectangle polygonal freehand 2 object cell membrane algorithm defined annotation in the field Note A ayer consists of objects cell membranes of a certain kind June 19 2009 3DHISTECH Ltd 9 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 About MembraneQuant 1 2 3 CCC files MembraneQuant saves Color Deconvolution settings into a CCC file that contains base settings by default 1 2 4 MISP files MembraneQuant algorithms are saved in MISP files that store the following information about the algorithm e segmentation algorithm Filters for noise reduction segmentation and object separation so that Mem braneQuant can isolate objects for example cell membranes on the digitized slide measurement parameters For example the diameters shorter longer the distance between two objects or the number of objects in an annotation 1 2 5 Using a MISP file on other slides You can reuse th
37. s Median Filter Local Intensity Amplifier Radius 2 Radius 25 Run 3 Threshold Separate M 24 E Seed distance gt 1 2 Filter Objects By Size l Filter Objects By Shape M Min 20 000 p Min 0 800 2 Max 100 000 Max 1 000 Y Restore DK Apply Cancel June 19 2009 3DHISTECH Ltd 26 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide Color Deconvolution Use this to set up the CD Color Deconvolution parameters Image Shows the mask and various aspects of the digitized slide Membrane Shows the various aspects of the membrane algorithm Smooth Blurring is recommended on an ordinary histology sample smoothness value is 3 by default and when working on low quality samples too much noise on the image it is recom mended to raise the value Merge membrane spots Membranes can be merged respect ively to their border intensity weakness length and area Change parameters until you get the desired detection result in the pre view window Membrane Settings Parameter settings of the membrane algorithm You can adjust the smoothness and the merging values Filter Sets the size in um and the average intensity of the objects that MembraneQuant should detect as objects There is a post pro cessing filter option in the algorithm based on Average intensity and Area values Thus the superfluous tissues gland lumens and cell cavities can be excluded from the detection process
38. t applications FISHQuant analyses FISH slides by detecting fluorescent spots and counter stained nuclei Evaluates the results based on the applied probes a Batch Processing Delete Measurements 2 Select the annotation or annotations you want to analyze in the Available win dow at the Annotations Selection panel then move them to the Selected window by clicking gt or gt gt If you need the whole slide check Process whole Slide Q Membrane G slides Membran 10301 Modify Membrane Image Segmentation Profile Definition Annotation Selection Process AnnotationArea Process whole Slide Available Selected Caption Rectangle Annotation Caption Freehand Annotation Export Settings C Save Region measurements C Save Cumulative measurements Membrane G slides Membran 10301 07_HER2_I Profile Setup Use this application to set or change the Profile Setup of the June 19 2009 3DHISTECH Ltd 23 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant MembraneQuant algorithm Annotation Selection You can select your annotation s for the measuring process Process Annotation Area The algorithm will be executed only on the selected annotations Process Whole Slide The annotation will be executed on the whole digital slide area Export settings ers Save Region measuremen
39. t specific MISP MIRAX Imaging Segmentation Profile A MembraneQuant segmentation and measuring algorithm is stored in a MISP file MRXS MIRAX Slide File extension of a digitized MIRAX slide Object A cell compartment for example membrane tissue cell or tissue that is relev ant for the immunohistochemical analysis Segmentation Identification of cells or cell compartments based on their different reactions to immunohistochemical staining June 19 2009 3DHISTECH Ltd 6 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 1 About MembraneQuant 1 About MembraneQuant MembraneQuant is an optional module for MIRAX Viewer It allows you to perform ex aminations of immunolabelled cell membrane on the virtual slides and digitalized with MIRAX microscopes MembraneQuant performs segmentation and measurements on MIRAX digital slides based on the colors of the immunoreactive cell membrane Based on the intensity of the immunohistochemical dyeing it can classify the cell membrane 1 1 MembraneQuant in the MIRAX product family The following picture shows how MembraneQuant and Measurement Visualization extend MIRAX Viewer and how they fit into the MIRAX product family Slides Fluorescent MIRAX Server slides wee TMA 1 MIRAX SCAN 2 Local file system 3 6 Image analysis modules 4 l el B Measurement Visualization 5 MIRAX Viewer 1 Slides are digitized with
40. ts The measurements will be expor ted to a CSV file that you can open and edit in any spreadsheet software for example MS Excel OpenOffice Save Cumulative measurements The measurements will be exported to CSV files A cumulative file will be created for each de tected ayer Save to single CSV The measurements will be exported to a single CSV file This file will contain all the parameters of the ay 3 Select Profile Setup to define the parameters of the algorithm The Membrane Analysis Setup window opens Color Deconvolution C Documents and Settings All Users Application Data 3D Membrane Detection Scoring Show results M Image Membrane fi Membrane Smooth 1 Z Merge membrane spots ner R Original v C No Nucleus Crossing Filter Objects By Size Min 120000 Mex 00 000 i HE When border intensity gt 200 Z Weakness Length Area pur lt g 100 00 s Filter s lt Average Intensity lt 50 Ja g lt Brea pur lt Z 20 00 800 00 Nucleus Median Filter Local Intensity Amplifier Radius 2 Z Radius Run ES Threshold r Separate 24 al 255 Seed distance gt 1 Filter Objects By Shape Min 0 800 S Max 1 000 iS June 19 2009 3DHISTECH Ltd 24 43 MembraneQuant for MIRAX Viewer 1 12 User s Guide 3 Working with MembraneQuant 4 Before setting the
Download Pdf Manuals
Related Search